Tyrosine separation from cystine

The hydrolysis of these compounds by the enzyme was determined by the isolation of the individual substances. The isolation of the amino acids soluble with difficulty in water, namely, tyrosine and cystine, presented no great difficulty, since those compounds crystallised out during the process of hydrolysis, but in the other cases the amino acids required separation from unchanged dipeptide. The ester method here again proved its usefulness the esters of the simple monoamino acids are easily volatile in vacuo and can be characterised by the methods previously described those of the dipeptides are not volatile and are characterised by conversion into their diketopiperazines or anhydrides by the action of ammonia, which compounds are less soluble than the dipeptides themselves and are thus capable of separation by filtration. 

FIGURE 10.15 Separation of 17-component amino acid hydrolyzate. Conditions Column, Dionex AS-8 gradient. Peaks (25 nmol each, except 12.5 nmol for cystine) a, arginine b, lysine c, threonine d, alanine e, glycine f, serine g, valine h, proline i, isoleucine j, leucine k, methionine 1, histidine m, phenylalanine n, glutamic acid o, aspartic acid p, cystine and q, tyrosine. (Reprinted from Welch, L.E., LaCourse, W.R., Mead, D.A., Jr., and Johnson, D.C., Ana/. Chem., 61, 555, 1989.)... 

The separation of cystine and tyrosine as they are obtained by hydrolysis with hydrochloric acid was described by Morner in I901. The protein—hair, keratin from horn, eggshells, etc.—was boiled with five times its quantity of 13 per cent hydrochloric acid under a reflux condenser on a water bath for six to seven days. The solution was then decolorised with charcoal and evaporated in vacuo, and the residue dissolved in 60 to 70 per cent, alcohol. The two acids then crystallised out on neutralising with soda, and were separated by fractional crystallisation from ammonia if much tyrosine was present it separated out first, but if cystine exceeded tyrosine in quantity this compound crystallised out first the remainder was only separated with difficulty. Embden separated the mixture of the two acids by means of very dilute nitric acid, in which tyrosine is very easily soluble, but cystine with difficulty. Their separation may also be effected by precipitation with mercuric sulphate in 5 per cent, sulphuric acid solution in which the mercury compound of tyrosine is soluble (Hopkins and Cole). 

The separation and estimation of the two main groups of amino acids can be carried out in one experiment, instead of separately as described. The protein is hydrolysed by sulphuric acid, the tyrosine, cystine and diaminotrioxydodecanic acid are removed by crystallisation, and the diamino acids are precipitated by phosphotungstic acid. From this precipitate they are obtained by decomposition with baryta, and they are then separated by means of their silver compounds by Kossel, Kustcher and Patten s method. The filtrate from the phosphotungstic acid precipitate is freed from the excess of phosphotungstic acid by means of baryta, and the solution is treated by Fischer s ester method for the monoamino acids. 

Figure 4 HPLC separation of 17 PTC AAs (Pico-Tag column 150 x 3.9mm, eluent A 0.14moll NaAc, pH 6.4, containing 0.5ml TEAI B 60% acetonitrile in water flow rate 1 ml minpeaks 1 = aspartic, 2=glutamic acids, 3 = serine, 4=glycine, 5=his-istidine, 6 = arginine, 7=threonine, 8 = alanine, 9 = proline, 10 = ammonia, l1=tyrosine, 12=valine, 13=methionine, 14=cystine, 15 = isoleucine, 16 = r>leucine, 17 = phenylalanine, 18=tryptophan. (Reproduced with permission from Bidlingmayer BA ef a/. (1984) Journal of Chromatography 336 Elsevier.)...

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